Paxgene blood rna system kit

Gene expression data was generated in the three cohorts separately using HumanHT-12 v4 Expression BeadChips (Illumina Inc., San Diego, CA, United States), which capture 47,231 expression probes. For the MIRECC/Duke and INTRuST samples, whole blood was collected in PAXgene blood RNA tubes and incubated at room temperature overnight before being transferred to −20°C for 24 h and finally stored at −80°C. Total RNA was extracted using the PAXgene Blood RNA System Kit following manufacturer’s guidelines (Qiagen, Germantown, MD, United States). Purified RNA was analyzed for integrity (RIN) using the Agilent Bioanalyzer 2100 with Agilent RNA 6000 LabChip kits (Agilent, Santa Clara, CA, United States) and only samples with RIN ≥ 6 were included in downstream analyses. All RNA samples were processed using the Ambion GLOBINclear-Human Globin mRNA Removal Kit to deplete alpha and beta globin mRNA and to increase sensitivity of gene detection (Life Technologies, Foster City, CA, United States). The enriched RNA was amplified and biotin-labeled using the Illumina TotalPrep Amplification Kit before hybridization to the microarrays. Details regarding the generation of gene expression data for the GMRF-QUT cohort have been described previously (Mehta et al., 2017 (link), 2018 (link)).

Maternal blood samples were collected at point of hospital admission prior to any medical treatment. Five PAXgene blood collection tubes (PreAnalytics, Hombrechtikon, Switzerland) were collected from each participant, total RNA was isolated using PAXgene Blood RNA system kit (QIAGEN, Doncaster, Victoria, Australia). RNA quality check was done at The Centre for Applied Genomics (TCAG; The Hospital for Sick Children (SickKids), Toronto, ON, Canada) using an Agilent 2100 BioAnalyser with the RNA 6000 Nano Kit (Agilent Technologies, Santa Clara, CA). The bioanalyser provides a RNA integrity number (RIN) to gauge RNA integrity, compare samples and ensure the repeatability of experiments. RIN is calculated using an algorithm and the bioanalyzer's electrophoretic trace where a RIN score of one represents strongly degraded RNA and a score of 10 represents intact RNA [28] (link). Adhering to TCAG's protocol, microarrays were only performed on samples with RIN greater than six.

RNA is extracted from whole blood using the PAXgene™ Blood RNA System Kit (Qiagen, Düsseldorf, Germany) employing an amended version of the manufacturer’s guidelines. Briefly, the samples are removed from −80 °C and incubated at room temperature for 2 h to ensure complete lysis. Following lysis, the tubes are centrifuged for 10 min at 5000× g (Boseo M-24 centrifuge, Hamburg, Germany), the supernatant is decanted, and 500 μL of RNase-free water is added to the pellet. The tube is vortexed to thoroughly resuspend the pellet, centrifuged for 10 min at 5000× g, and the entire supernatant discarded. The remaining pelleted lysate is re-suspended in 360 μL of buffer BR1 by vortexing, and the manufacturer’s protocol is followed from this step [14 (link),15 ].
Freshly extracted RNA is measured using a NanoDrop ND-1000 UV-visible spectrophotometer (Thermo Fisher, Waltham, MA, USA). The software displays the concentration in ng/μL. There is also a quality output, which provides 260/280 and 260/230 ratios enabling purity estimations. RNA integrity is additionally assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), and poor quality samples are excluded from the analysis [15 ].